Costimulatory genes and molecules have a profound impact on autoimmune diseases, as shown by a number of experiments in" animal models using mice that are deficient in key costimulatory molecules, or by blocking the molecules or their ligands in vivo. On the other hand, it has been difficult to understand and compare relative roles of each of costimulatory molecules in different autoimmune disease models since apparently conflicting observations have been made. This may be ascribed to true differences in requirement for costimulation between the autoimmune diseases studied (in particular Experimental Autoimmune Encephalomyelitis - EAE - and Type-1 Diabetes - T1D), or to differences in reagents used in each experimental setting. Further, there is growing evidence that costimulatory molecules, and the relative balance of their effects, vary among inbred mouse strains. Some of the differences may thus reflect different importance of particular molecules in different inbred backgrounds. Finally, it is possible that some of the effects ascribed to one costimulatory molecule may be indirect, since loss of one costimulatory molecule may affect upregulation of other costimulatory molecules. Using existing mutant mouse strains and additional resources created in this program project, we propose to resolve these issues and address how inheritance of haplotype controls differences in costimulation on different backgrounds and different autoimmune disease models: 1. Analyze in parallel the parameters of EAE and T1D in the same mice, carrying single mutations in costimulatory molecules (CD28, ICOS), and/or complemented by constitutive transgene-driven expression of other family members. These will be pursued on both C57BV6 (B6) and NOD backgrounds, to ascertain the effects of genetic polymorphism. 2. We have recently discovered a novel form of CTLA4, which may have dominant influence on the activity of CTLA4 or other costimulatory molecules. It is expressed at different levels in B6 and NOD mice. We will explore its function, and its impact on EAE and T1D development, utilizing a novel transgenic strain in which this variant is constitutively expressed. 3. Hypothezing that genetic variants of linked CD28/ICOS/CTLA4 costimulatory genes may segregate as balanced haplotypes, we will investigate more extensively the genetic polymorphism of the loci in a panel of inbred strains. We will correlate the genetic variation with the known functional polymorphism in the loci, identify recombinant chromosomes separating the B6 and NOD alleles. Parallel investigations will be performed in the human genome, searching for concerted transmission of "haplotypic blocs" in the human genome. Together, these data should illuminate the comparative importance of costimulatory molecules in the genesis of EAE and T1D, their regulatory interplay, and how their known genetic and functional variations are transmitted in the murine and human species.